Journal of Steroid Biochemistry & Molecular Biology 112 (2008) 110–116

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Journal of Steroid Biochemistry and Molecular Biology

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Species difference exists in the effects of 1�,25(OH)2D3 and its analogue2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD)on osteoblastic cells

Yan Lia,∗, Carl-Magnus Bäckesjöa, Lars-Arne Haldosénb, Urban Lindgrena

a Department for Clinical Science, Intervention and Technology (CLINTEC), Division of Orthopedics, Karolinska Institutet, 141 86 Huddinge, Swedenb Department of Biosciences and Nutrition, Karolinska Institutet, Novum, 141 86 Huddinge, Sweden

a r t i c l e i n f o

Article history:Received 25 October 2007Received in revised form 17 July 2008Accepted 2 September 2008

Keywords:2MD1�,25(OH)2D3

Osteoblast

a b s t r a c t

The direct effect of 1�,25(OH)2D3 on osteoblasts remains unclear. In this study, we evaluated the in vitroeffects of 1�,25(OH)2D3 and its analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD),on osteoblasts from three different species, i.e. bone marrow stromal cells from the Sprague–Dawley(SD) rat, from the C57BL/6 mouse, as well as human osteoblast NHOst cells and human osteosarcomaderived MG-63 cells. We found that in rat cells, both compounds increased cell proliferation, inhibitedcell apoptosis and increased alkaline phosphatase (ALP) activity. In mouse cells, however, both compoundsinitiated cell apoptosis and inhibited ALP activity. In human cells, although cell proliferation was inhibitedby both compounds, cell apoptosis was inhibited and ALP activity was enhanced. In each species, 2MD was

ApoptosisAlkaline phosphatase (ALP)

much more potent than 1�,25(OH)2D3. To summarize, species differences should be taken into account instudies of vitamin D effects. However, in all tested species – rat, mouse and human – 2MD is considerably

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. Introduction

The physiologically active form of vitamin D, 1�,25(OH)2D3,s a seco-steroid hormone. Its effects are mediated primarily viahe vitamin D receptor (VDR), which is a member of the nucleareceptor superfamily. When bound by its ligands, the VDR dimer-zes with the retinoic X receptor (RXR) and binds to promoteregions of responsive genes to either activate or repress tran-cription [1,2]. It is generally agreed that 1�,25(OH)2D3 affectsone formation mainly by indirect mechanisms on calcium home-stasis [2]. Direct effects on osteoblasts have been reported buthe findings have sometimes been contradictory, for example,�,25(OH)2D3 has been shown to increase the production of osteo-alcin and alkaline phosphatase (ALP) in rat and human osteoblasts3,4], while in mouse osteoblasts 1�,25(OH)2D3 down-regulated

xpression of Phex, a mature osteoblast marker, and blocked initro mineralization [5,6]. We felt that the role of species dif-erences in this controversy needed to be clarified further. As

synthetic 1�,25(OH)2D3 analogue, 2-methylene-19-nor-(20S)-�,25(OH)2D3 (2MD) differs structurally from 1�,25(OH)2D3 by

∗ Corresponding author. Tel.: +46 8 58583869; fax: +46 8 58582224.E-mail address: Yan.Li@ki.se (Y. Li).

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teoblastic cells in vitro than 1�,25(OH)2D3.© 2008 Elsevier Ltd. All rights reserved.

he absence of the 19-methylene carbon and the addition of aethylene group in the 2-position between the 3�-hydroxyl and

�-hydroxyl. In addition, the C-20 configuration is changed to0S compared with a 20R configuration in naturally occurringitamin D compounds [7]. In rat studies, 2MD stimulated boneormation without triggering noticeable hypercalcemia and hyper-alciuria; additionally, although 2MD was about 30–100-fold moreotent than 1�,25(OH)2D3 in bone calcium mobilization it waso more effective in promoting intestinal calcium absorption [8].urther studies in ovariectomized rats showed that low dosesf 2MD increased total bone mass and promoted the synthesisf both trabecular and cortical bone with high quality, whereasuch higher doses of 1�,25(OH)2D3 only prevented bone loss

ver the same period [9]. Such bone anabolic effects imply thatMD increases bone formation through a direct action on boneells.

The bone forming osteoblasts originate from pluripotent mes-nchymal stem cells [10]. After maturing and participating in boneemodeling most of the cells undergo apoptosis [11,12]. In theresent study, we used osteoblastic cells from different species,

.e. bone marrow stromal cells from Sprague–Dawley (SD) ratnd C57BL/6 mouse, as well as human osteoblast NHOst andsteosarcoma MG-63 cells. We compared herein the effects of�,25(OH)2D3 and 2MD on cell proliferation, apoptosis and alkalinehosphatase activity.

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. Materials and methods

.1. Compounds

2MD was synthesized by Tetrionics (Madison, WI) by meth-ds previously described [7] and was kindly supplied by Dr. HFeLuca. 1�,25(OH)2D3 was purchased from Calbiochem (Darm-

tadt, Germany). Both reagents were diluted in 99% ethanol in stockoncentrations for experimental use.

.2. Cell culture

Rat bone marrow stromal cells were obtained from 6-week-oldale Sprague–Dawley rats. Briefly, rats were euthanized using 4%

sofluorane in CO2, and the bones were aseptically excised from theindlimbs. External soft tissue was discarded and the bones werelaced in 50 ml alpha-MEM (Invitrogen, Life Technologies, Paisley,cotland) supplemented with 400 �g/ml of gentamicin (Invitrogen,ife Technologies, Paisley, Scotland). Both ends of the femur andibia were clipped. An 18-gauge needle was inserted into the holen one end and bone marrow was flushed out from the other to a0 ml Falcon tube by culture medium, i.e. alpha-MEM medium sup-lemented with 10% fetal bovine serum (FBS), 1 mM l-glutaminend 100 �g/ml gentamicin. After centrifugation at 1000 rpm formin, the cell pellet was collected and diluted in 15 ml cultureedium and cultured in a 75 ml flask. Nonadherent cells were

emoved after 24 h and the remaining cells were split after reaching0% confluence. Mouse bone marrow stromal cells were harvestedrom 2-week-old male C57BL/6 mice by the same procedure. Nor-

al human osteoblast cell line NHOst was obtained from Cambrexnd cultured in Osteoblast Growth Media (OGM) supplemented

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ig. 1. Effects of VDR ligands on proliferation of osteoblastic cells. Cells were cultured in 96-f 1�,25(OH)2D3 or 2MD. Cell proliferation was measured on day 2, day 4 and day 8 byone marrow stromal cells. (B) Mouse bone marrow stromal cells. (C) Human osteosarcomehicle control on the same day; #P < 0.05 compared with vehicle control on the same day

Molecular Biology 112 (2008) 110–116 111

ith OGM SingleQuots. Human osteosarcoma cell line MG-63 wasbtained from American Type Culture Collection and cultured in �-EM medium supplemented with 10% FBS, 1 mM l-glutamine and

00 �g/ml gentamicin. All cells were cultured at 37 ◦C in a humid-fied atmosphere containing 5% CO2. For inducing differentiationowards osteoblast lineage, the culture medium was added with0 mM �-glycerophosphate, 100 �g/ml ascorbic acid and 0.1 mMexamethasone (bone inducing medium).

.3. Cell number measurement

Cells were seeded at 5000 cells per well in 96-well plates andultured in 100 �l bone inducing medium containing vehicle orifferent concentrations of 1�,25(OH)2D3 or 2MD (as indicated inig. 1). The medium was renewed every other day and several non-ell plated wells were also renewed by medium to serve as blankontrols. Total cell amounts were measured on day 2, day 4 and dayusing the following procedure: 10 �l cell proliferation reagentST-1 (Roche Applied Science, Stockholm, Sweden) was added to

ach well and gently mixed with the medium. After incubationt 37 ◦C for 40 min the plates were read by a kinetic ELISA readerSpectra MAX 250, Molecular Devices, CA, USA). Cell amount wasndicated by absorbance at wavelength of 450 nm with subtractionf background absorbance at reference wavelength 650 nm. At leastour wells were used for each concentration of the tested reagents.

.4. Flow cytometric analysis of apoptosis

For flow cytometric analysis of apoptosis, cells were plated at00,000 per well in 6-well plates. After 24 h, the medium washanged to serum-reduced, i.e. 2.5% FBS, bone inducing medium

well plates in bone inducing medium containing vehicle or different concentrationsWST assay. Each data point represents the mean ± S.E.M. of four samples. (A) Rata MG-63 cells. (D) Normal human osteoblast NHOst cells. (*P < 0.01 compared with.)

112 Y. Li et al. / Journal of Steroid Biochemistry & Molecular Biology 112 (2008) 110–116

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ig. 2. Effects of VDR ligands on apoptosis of osteoblastic cells. Cells were culturedr 1 nM 2MD. Cell apoptosis was analyzed on day 3 and day 9 by flow cytometry. Eacells. (B) Mouse bone marrow stromal cells. (C) Human osteosarcoma MG-63 cells.

ontaining vehicle, 1 nM 1�,25(OH)2D3 or 1 nM 2MD. Cells wereultured for 3 and 9 days. At harvest, adherent cells were digestedy trypsin and combined with floating cells in the medium. Allells were centrifuged and washed with PBS. Twenty thousandells were suspended in 100 �l apoptosis binding buffer, and thentained by Annexin-FITC and/or propium iodide using an Annexinkit (Caltag Laboratories, Burlingame, CA, USA). Positive controls

or apoptosis were obtained by adding 100 nM staurosporine onontrol cells 4 h before staining with only Annexin-FITC. Positiveontrols for necrosis were obtained by fixing control cells in −20 ◦C0% ethanol for 20 min before staining with only propium iodide.t least 10,000 cells were analyzed by flow cytometry using aACScan-equipment and CellQuest software (Becton Dickson Co.,ranklin Lakes, NJ, USA). The cells binding Annexin V were recordeds apoptotic cells, cells binding propium iodide only were recordeds necrotic cells and double negative cells were recorded as livingells. Cell number in each group was divided by the number of totalated cells and the percentages were used for statistical analysis.t least three wells were used for each tested reagent.

.5. Quantification of alkaline phosphatase activity

Phosphatase Substrate Kit (Pierce, IL, USA) containing PNPP (p-itrophenyl phosphate disodium salt) was used to quantify thelkaline phosphatase activity in cell cultures. Cells were platedt 5000 cells per well in 96-well plates and cultured in bonenducing medium containing vehicle or different concentrations of

�,25(OH)2D3 or 2MD (as indicated in Fig. 3). Alkaline phosphatasectivity was measured after 4 days of culture, according to the fol-owing protocol: PNPP solution was prepared by dissolving twoNPP tablets in 8 ml of distilled water and 2 ml of diethanolamineubstrate buffer. After washing twice with PBS, cells were incubated

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point represents the mean ± S.E.M. of three samples. (A) Rat bone marrow stromalrmal human osteoblast NHOst cells.

ith 100 �l/well PNPP solution for 30 min at room temperature.ifty microliters of 2N NaOH was added to each well to stop theeaction. Non-cell plated wells treated by the same procedure weresed as blank control. The absorbance was measured at 405 nm inkinetic ELISA reader (Spectra MAX 250, Molecular Devices, CA,SA). At least four wells were used for each concentration of the

ested reagents.

.6. Alkaline phosphatase staining

TRACP & ALP double-stain kit (Kakara Bio. Inc., Otsu, Japan) wassed for staining of alkaline phosphatase in the cell cultures. Cellsere plated at 50,000 cells per well in 24-well plates and grown

or 4 days in bone inducing medium containing vehicle, differ-nt concentrations of 2MD and 1�,25(OH)2D3. Substrate for ALPas dissolved in 10 ml of distilled water before use. After wash-

ng twice with PBS cells were fixed in fixation buffer for 5 min.fter three times washes with distilled water, 250 �l of ALP sub-trate was added to each well. Cells were incubated at 37 ◦C for0 min. After washing again with distilled water, the cells werebserved and photographed by an inverted fluorescence micro-cope system (Zeiss Axiovert S100, Spectral Solutions, Stockholm,weden).

. Results

.1. Effects of 1,25(OH)2D3 and 2MD on growth of osteoblastic

ells

In order to investigate the effects of 1�,25(OH)2D3 and 2MDn proliferation of osteoblastic cells, we cultured the cells in bonenducing medium for 8 days in the presence of different con-

Y. Li et al. / Journal of Steroid Biochemistry & Molecular Biology 112 (2008) 110–116 113

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ig. 3. Effects of VDR ligands on alkaline phosphatase (ALP) activity of osteoblastic cifferent concentrations of 1�,25(OH)2D3 or 2MD for 4 days. Phosphatase Substrateo quantify the alkaline phosphatase activity in cell cultures. At least four wells weren each well; right part, ALP activity after normalization by proliferation. (A) Rat bon

G-63 cells. (D) Normal human osteoblast NHOst cells. (*P < 0.01 compared with ve

entrations of the two VDR-ligands. In rat cells (Fig. 1A), both�,25(OH)2D3 and 2MD stimulated cell growth most apparentlyn day 8. However, in mouse cells (Fig. 1B), the results were appar-ntly opposite to that in rat cells with all tested doses of 2MD, as

ell as the highest dose of 1�,25(OH)2D3 which elicited potent

nti-proliferative effects. The effects on human osteoblastic cellsere similar to the seen with mouse cells, i.e. 2MD appeared to beore potent than 1�,25(OH)2D3 in inhibiting cell growth of MG-63

nd NHOst cells (Fig. 1C and D).

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ells were cultured in 96-well plates in bone inducing medium containing vehicle orierce, IL, USA) containing PNPP (p-nitrophenyl phosphate disodium salt) was used

for each concentration of the tested reagents. Left part, total ALP activity of the cellsrow stromal cells. (B) Mouse bone marrow stromal cells. (C) Human osteosarcomaontrol on the same day; #P < 0.05 compared with vehicle control on the same day.)

.2. Effect of 1,25(OH)2D3 and 2MD on apoptosis of osteoblasticells

Cell number is influenced by both mitosis and cell death. In this

xperiment, we cultured the cells in serum-reduced bone inducingedium (2.5% FBS) up to 9 days. As indicated in Fig. 2, with-

ut treatment with VDR ligands the percentage of living cells inhe four different cell types decreased from day 3 to day 9 to aariable extent. In rat cells (Fig. 2A), both VDR ligands showed sig-

114 Y. Li et al. / Journal of Steroid Biochemistry & Molecular Biology 112 (2008) 110–116

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ig. 4. Staining of alkaline phosphatase (ALP) in osteoblastic cells. Cells were cultuoncentrations of 2MD or 1�,25(OH)2D3. TRACP & ALP double-stain kit (Kakara Bio. Int 10 × 20 magnification with an inverted fluorescence microscope system (Zeiss AxB) Mouse bone marrow stromal cells. (C) Human osteosarcoma MG-63 cells. (D) N

ificant anti-apoptotic effects as measured on day 9 (53.19 ± 3.59%ells were living in the control group while 72.09 ± 2.62% wereiving in the 1�,25(OH)2D3 group and 84.39 ± 1.72% in the 2MDroup). However, in mouse cell (Fig. 2B), both VDR ligands stronglynduced apoptosis at both time points (88.44 ± 0.90% of total cells

ere living in the control group, 81.90 ± 2.90% were living in the�,25(OH)2D3 group and 44.61 ± 2.14% in the 2MD group on day; on day 9, 75.87 ± 1.30% of cells were living in the control group,5.22 ± 1.79% and 21.95 ± 3.05% were living in the 1�,25(OH)2D3nd 2MD group, respectively). Interestingly, unlike the results fromroliferation assay, both human osteoblastic cell lines exerted sim-

lar responses to the VDR ligands as the rat cells, i.e. significantnti-apoptotic effects were found on day 9 of culture. In MG-3 cells, 46.44 ± 2.33% of total cells were living in the controlroup, 70.70 ± 4.87% were living in the 1�,25(OH)2D3 group and6.27 ± 2.19% in 2MD; in NHOst cells, 77.54 ± 1.01% of cells were

iving in the control group, 82.44 ± 0.18% and 85.96 ± 0.56% wereiving in the 1�,25(OH)2D3 and 2MD groups, respectively. Addi-ionally, in all tested cell types the effect of 2MD was more potenthan 1�,25(OH)2D3.

.3. Effects of 1˛,25(OH)2D3 and 2MD on alkaline phosphatase

ctivity in osteoblastic cells

Bone formation is affected not only by the amount of osteoblastsut also by their functional activity. In order to investigate theffects of the VDR ligands on osteoblast function, we measured

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t S100, Spectral Solutions, Stockholm, Sweden). (A) Rat bone marrow stromal cells.human osteoblast NHOst cells.

lkaline phosphatase activity in cells treated with different con-entrations of each VDR ligand. Fig. 3 shows the results from theemi-quantitative analysis of ALP activity by PNPP methods. Theesults in the left part show the total ALP activity of the cells inach well. In rat bone marrow stromal cells (Fig. 3A), 1�,25(OH)2D3ignificantly increased ALP activity at 1 nM and the effect becameore significant as the concentration increased to 10 nM. With

MD a significant increase of ALP activity has already observed at.01 nM. In mouse cells (Fig. 3B), however, 1�,25(OH)2D3 signif-

cantly inhibited ALP activity at concentrations higher than 1 nMnd 2MD almost totally blocked ALP activity at any tested con-entration. As indicated in Fig. 3C and D, in human osteoblasticells both VDR compounds increased ALP activity. In MG-63 cellsFig. 3C), 1�,25(OH)2D3 increased ALP activity dose-dependentlyrom 0.1 nM while 2MD exerted a comparable effect from 0.01 nM.n NHOst cells, significant increase of ALP activity was elicited by0 nM of 1�,25(OH)2D3 and all dosages of 2MD. The total ALPctivity of the cells in each well could also be affected by cellmount. Thus, in order to investigate the effects of the VDR lig-nds on individual cells we normalized the ALP data to the cellumber for each concentration of the tested compounds. As indi-ated in the right part of each figure, similar patterns were found

hich show that both VDR ligands increased the ALP activity in

at and human cells but inhibited ALP activity in mouse cells. Fur-hermore, in each species the effect of 2MD was more potent than�,25(OH)2D3. The results of PNPP analysis were further confirmedy ALP staining (Fig. 4). In rat osteoblasts (Fig. 4A), only a few

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ells showed positive staining in the control group. 1�,25(OH)2D3ncreased the number of positively stained cells apparently at 10 nMosage while similar effects could be elicited by 2MD at 0.01 nM.or cells treated with 0.1 and 1 nM 2MD even more ALP positiveells were seen. In mouse cells (Fig. 4B), many cells were stainedositively in the control group. The positive staining was apparentlyeduced with 1 nM 1�,25(OH)2D3 and almost totally disappearedt the 10 nM dosage. Consistent with PNPP measurement, almosto mouse cells were positively stained with the treatment of 2MDt any tested concentration. For human osteosarcoma MG-63 cellsFig. 4C), few positive-stained cells were seen with the treatmentf vehicle as well as with 0.1 and 1 nM 1�,25(OH)2D3. Ten nanomo-ars of 1�,25(OH)2D3 apparently increased the number of positivetained cells. With 2MD the increase of positive stained cells coulde already noticed at 0.01 nM. For human osteoblast NHOst cellsFig. 4D), an increase of positive staining could be seen at all testedoncentrations of 2MD. With 1�,25(OH)2D3 no increase of stainingould be seen, even at the highest dose tested, 10 nM.

. Discussion

In the present study osteoblastic cells from different species,at, mouse and human were evaluated regarding the responses towo VDR ligands, 1�,25(OH)2D3 and 2MD. Our study focused onwo important issues about the direct effects of VDR ligands onsteoblasts: the species difference and the comparison betweenMD and 1�,25(OH)2D3.

Cells from different species respond to VDR ligands differently.he rat is the most commonly used animal for vitamin D studies.imilar to humans, rats respond to the treatment of 1�,25(OH)2D3ith increase in intestinal calcium absorption and bone calciumobilization [13]. Rat experiments indicate that 1�,25(OH)2D3

ot only induces osteoblast differentiation and bone formationn vitro [3,4] but also prevents experimental osteoporosis in vivo14–19]. VDR-knockout mice show typical features of vitamin D-ependent rickets type II, such as failing to thrive after weaning,ppearance of alopoecia, hypocalcaemia and infertility, as well aseverely impaired bone formation [20]. However, studies from VDRnockout mice did not support any direct effects of 1�,25(OH)2D3n bone formation and rickets can be totally prevented by highalcium diet alone [21]. Furthermore, transplantation of bonerom VDR knockout mice to wild-type mice revealed a significantncrease in bone volume and density compared with control (wild-ype bone transplanted to wild-type mouse), which indicated that�,25(OH)2D3 suppressed bone formation [22]. Undesirable effectsf 1�,25(OH)2D3 have also been reported in other species, such asn rabbits, in which 1�,25(OH)2D3 prevented fracture healing andggravated immobilization or prednisolone-induced osteoporosis23]. Previously, the reason for these paradoxes was supposed to behe difference in the calcium metabolism mechanism [23], how-ver, no further evidence has been reported. Our results showhat both VDR ligands increased cell number and ALP activity inat osteoblasts, while they reduced cell population and inhibitedLP activity in mouse cells. Additionally, regarding the humansteoblastic cells, although cell growth was inhibited by VDR lig-nds ALP activity was enhanced and apoptosis was prevented.hese results are consistent with in vivo findings in rats and VDRnockout mice. Thus, the different effects of 1�,25(OH)2D3 on boneormation among different species are probably influenced by its

irect effects on osteoblasts. The transcriptional and posttranscrip-ional control of gene expression is mediated by 1�,25(OH)2D3 on

ultiple levels. Therefore, the molecular mechanisms contributingo the species differences are likely to be complex. A nucleotideequence variation between rat and mouse osteoblasts has been

Molecular Biology 112 (2008) 110–116 115

ound in the distal half motif of osteocalcin VDRE and contributedignificantly to their different response to 1�,25(OH)2D3 [24]. Ouresults indicate that such nucleotide sequence variations in VDRErobably exist for many other important genes which control cellrowth and activity and thus determine the different response ofhe osteoblasts from different species to VDR ligands.

The bone anabolic effects of 2MD in rats are probably medi-ted by its direct effects on osteoblasts. Although 1�,25(OH)2D3an decrease bone loss in ovariectomized rats [14,19] and mightecrease fracture rates in osteoporotic patients [25] there is lim-

ted evidence that the hormone can actually increase bone mass.MD, however, has been shown to not only induce mineraliza-ion in vitro but also increase bone density in ovariectomized ratsn vivo [8]. This makes 2MD a VDR ligand to study further. Ouresults showed that both 2MD and 1�,25(OH)2D3 increased ratsteoblast cell number and 2MD demonstrated a much strongernti-apoptotic effect than 1�,25(OH)2D3. We have previouslyhown that 1�,25(OH)2D3 elicited anti-apoptotic effects on ratsteoblast-like osteosarcoma UMR 106 cells by increasing the Bcl-/Bax ratio and by inhibiting caspase-3 activity [26]. Whether 2MD

nhibited cell apoptosis through the same mechanism and how theffects are enhanced by 2MD need to be further clarified. Addition-lly, we found that 2MD was much more potent than 1�,25(OH)2D3n increasing ALP activity in rat osteoblasts. ALP plays an impor-ant role in facilitating bone mineralization through mechanismsuch as breakdown of the major mineralization inhibitor, inorganicyrophosphate [27]. The combination effects of inhibiting apopto-is and enhancing ALP activity in osteoblasts may result in a largermount of osteoblasts which can form more organic bone matrix inmicroenvironment favorable of bone mineralization. These spe-

ific effects on osteoblasts, together with its moderate effects innhancing intestinal calcium absorption, could be the explanationor the dramatic bone anabolic effects of 2MD. However, despite ofhe higher potency of 2MD compared with 1�,25(OH)2D3 we didot find any qualitative difference between the two VDR ligandsn osteoblasts. It is possible that the effects of 2MD on osteoblastsould be elicited by a higher dose of 1�,25(OH)2D3 but in vivo thisould entail a high risk of hypercalcemia. On the other hand, inuman osteoblastic cells, although 2MD dramatically increased ALPctivity the cell number was actually reduced. Thus, more human-ased experiments are needed in order to confirm the potential ofMD as an agent used for treating osteoporosis.

In conclusion, in the present study species difference regard-ng the direct effects of two VDR ligands on osteoblastic cells washown. In each species, 2MD demonstrated a much higher potencyhan its native analogue, 1�,25(OH)2D3. The capacity of 2MD innhibiting osteoblast apoptosis and increasing ALP activity coulde the main reason for its known bone anabolic effects in rats.

cknowledgements

Åsa-Lena Dackland is gratefully acknowledged for technical sup-ort with the FACScan equipment. We thank Hector De Luca forupplying us with 2MD. This study was supported by the Karolinskanstitute and the Swedish Foundation for Strategic Research.

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